The present invention relates to novel haptotactic peptides, and in particular, to novel peptides which are homologous to a portion of the carboxy termini of fibrinogen chains, as well as to potential uses for these peptides.
Fibrinogen is the plasma protein which forms the clot when blood coagulates. Many studies have been conducted on the amino acid sequences and structure of fibrinogen (Mosesson, M. and Doolittle, R. (Eds.) “The biology of fibrinogen and fibrin”, Ann. N.Y. Acad. Sci., 408, 1983, Henschen, A. et al., “Structure of fibrinogen”, Ann. N.Y. Acad. Sci., 408, 1983, Spraggon, G. et al., “Crystal structure of fragment D from human fibrinogen and its crosslinked counterpart from fibrin”, Nature. 389:455–462, 1997, Murakawa, M. et al., “Diversity of primary structures of the carboxy-terminal regions of mammalian fibrinogen Aα-chains”, Thromb. & Haemostat., 69:351–360, 1993). Normally, fibrinogen itself has a molecular weight of 340 kDa and is constructed from two sets of three peptide chains, named α, β, and γ. The constituent chains of fibrinogen are highly conserved between species. Recent work has also described a fibrinogen protein with a longer α chain, called αE fibrinogen, which has a concomitantly higher molecular weight of 420 kDa and which may play a role in development (Fu, Y. and Grieninger, G. “Fib420: A normal human variant of fibrinogen with two extended α chains”. Proc. Natl. Acad. Sci. USA. 91: 2625–2628, (1994), Fu, Y. et al., “Carboxy-terminal-extended variant of the human fibrinogen α subunit: A novel exon conferring marked homology to β and γ subunits” Biochem., 31:11968–11972, (1992)). Thus, these four types of fibrinogen chains, α, β, γ and αE, have 610, 410, 391 and 1096 amino acids, respectively (numbering based on the Gene-bank data base at info@ncbi.nlm.nih.gov).
Fibrin clots are formed in vivo at the sites of tissue injury based upon the reaction of fibrinogen and thrombin in the presence of calcium ions. These clots have a major role in hemostasis. After clot formation, fibrin serves a provisional matrix for cell recruitment into the wound bed. Normally, the earliest cells mobilized into the wound bed are inflammatory, such as leukocytes and particularly macrophages. Concomitant With their penetration into the fibrin, these inflammatory cells participate in lysing the fibrin by generating plasmin, metallo-proteinases (MIPs) and/or free radicals. Thus, the wound bed contains substantial quantities of peptides A and B (FPA and FPB) released by thrombin during the onset of coagulation and numerous fibrin breakdown products are generated by lytic enzymes or free-radicals (Gray, A. J., Reeves, J. T., Harrison, N. K., Winlove, P. and Laurent, G. J., “Growth factors for human fibroblasts in the solute remaining after clot formation”, J. Cell Sci., 96: 271–274, (1990), Marx G. “Immunological monitoring of Fenton fragmentation of fibrinogen”. Free Radicals Res. Comm. 12: 517–520 (1991), Francis, C. W., Marder, V. J. and Barlow, G. H., “Plasmic degradation of crosslinked fibrin”. J. Clin Invest., 66: 1033–1043, (1980), Cottrell, B. A. and Doolittle, R. F. “The amino acid sequence of a 27-residue peptide released from α-chain carboxy-terminus during the plasmic digestion of human fibrinogen”, Acad. Press., 71: 754–766, (1976)).
Subsequently, the inflammatory cells are followed by the migration of cells of the mesenchymal cell lineage such as fibroblasts which further digest fibrin, replacing it with extracellular matrix (ECM). Endothelial cells also infiltrate the wound bed and generate microcapillary structures. Ultimately these cells replace the provisional fibrin matrix with granulation tissue populated by parenchymal cells and vasculature within the newly synthesized ECM.
The attachment and migratory responses of cells to matrix were proposed to be controlled mainly by specific receptors (integrins) or by intercellular adhesion molecules (ICAM) that interact with cell membrane receptors which subsequently induce either migratory reactions or cell adhesion to matrix. These interactions may trigger other regulatory mechanisms of cell activity, such as shape change or proliferation. Growth factors and cytokines activate such cell receptors by binding to them, and thus, trigger cellular responses (Ruslahti, E. (1996) RGD and other recognition sequences for integrins. Ann. Rev. Cell Dev. Biol. 12, 697–715 and Hynes, R. O. (1992) Integrins: Versatility, modulation and signaling in cell adhesion. Cell 69, 11–25).
Cytokines of different classes regulate cellular activity and responses, control cell survival, growth and differentiation. Excluding classical endocrine hormones, cytokines encompass those families of cell regulators variously known as growth factors, interleukins, lymphokines and interferons.
All previously described cytokines are composed of more than 50 amino acids (aa); most are over 100 aa long. Based on X-ray crystallography, cytokines exhibit 8 structural groups (Nathan C. & Sporn M., “Cytokines in context”, J. Cell. Biol. 113: 981–986 (1991)) and bind to a variety of cellular receptors such as integrins or interferon receptors. Binding to cell receptors triggers a cascade of events leading to intra-cellular phosphorylation of proteins, which is transduced into gene expression, cell proliferation, cell differentiation, changes in cell shape, motility and apoptosis. Thus, cytokines play an important role in physiological processes such as development and wound healing.
Human fibroblasts are the major cellular entities responsible for the regeneration of the extracellular matrix Within the wound bed. Human fibroblasts also express specific membrane receptors to fibrinogen and thrombin. In the case of skin wounds, human fibroblasts reform the matrix of the dermis. For example, during the course of healing of an incisional skin wound, human fibroblasts are mobilized from the surrounding tissue and enter into the fibrin clot, help to dissolve the clot, and then generate as well as reform the collagen in the extracellular matrix. Based upon these properties of human fibroblasts, fibroblast implants have been suggested to supplement the process of healing in damaged skin (Gorodetsky, R. et al., Radiat. Res. 125:181–186, 1991).
One material used for this purpose is benzoylated hyaluronic acid (HA) sheets containing holes or pores as a carrier for fibroblasts and keratinocytes for wound healing (Andreassi, L., et al., Wounds, 3:116–126, 1991). Specifically, HA sheets were cultured with such cells which grow within the pore structure. The HA sheets were then affixed to the site of the burn injury, where the cells migrated out of the sheet and ultimately accelerated the rate of wound re-granulation. A major problem with implanted HA sheets, however, is that they are not metabolized by tissue, are mechanically cumbersome to administer, and may cause undesired immunological effects in the long term.
Another material used for prosthetic tissue engineering is collagen from pig or beef sources. However, collagen has several mechanical limitations and may reduce the new collagen synthesis by cells that are incorporated in it. There is also concern regarding the safety of animal collagen products for medical implantation and its use has been severely limited in Europe.
Fibrin microbeads (FMB) have been disclosed as possessing both chemotactic and proliferative effects for certain types of cells in U.S. application Ser. No. 08/934,283, filed in Sep. 19, 1997 and Gorodetsky, R., Vexler A., Shamir M., An J., Levdansky L., and Marx G. (1999). J. Invest. Dermatol. 112, 866–872 (1999)). The cells that are attracted to FMB include fibroblasts and smooth muscle endothelial cells, but typically not keratinocytes. The cells were shown to migrate into these FMB by chemotaxis, attach to them (haptotaxis) and then to proliferate on the FMB. Furthermore, the cells were shown to remain stable for prolonged periods of time when cultured within the FMB. Thus, the disclosed FMB appeared to stimulate both cell chemotaxis, haptotaxis and cell growth.
However, the fibrin microbeads themselves have certain inherent limitations. For example, the FMB are particularly useful only as three-dimensional micro-structures. If other structures were desired, and in particular if the lack of such was desired, FMB would not be particularly useful Furthermore, FMB would not be particularly useful for avoiding the use of blood plasma proteins.
A more useful approach would identify the epitopes of fibrin(ogen) responsible for its chemotactic and haptotactic properties. Attempts have been made to find these small epitopes within the larger fibrin(ogen) molecule. A voluminous literature exists which describes the binding of fibrinogen (γ400–411) to platelets through the GPIIb/IIa receptor (see for example Savage B., Bottini E. & Ruggeri Z M., “Interaction of integrin alpha IIb beta with multiple fibrinogen domains during platelet adhesion”, J. Biol. Chem. 270: 28812–7 (1995)), and the aggregation activity of the amino Bβ 15–42 terminus which is exposed after release of fibrinopeptide B. In addition, a peptide containing the 16 amino acids of the sequence of the γ-carboxy terminus of fibrinogen was synthesized and was found to bind to platelet integrin (D'Souza, S. E. et al., J. Biol. Chem., 265:3440–3446, 1990). However, the biological activities of only a few other fibrinogen breakdown products have been investigated with cells and the activity of these different breakdown products seems to be widely variable.
In another example, fibrinogen fragment E was reported to exhibit angiogenic properties and to inhibit endothelial cell migration in a Boyden chamber chemotactic assay (Thompson, W. D., Smith, E. B., Stirk, C. M., Marshall, F. I., Stout, A. J. and Kocchar, A., “Angiogenic activity of fibrin degradation products is located in fibrin fragment E”, J. Pathol. 168: 47–53 (1992)). Fragment D was reported to cause detachment of cultured endothelial cells from the extracellular matrix (ECM) substratum in a process which was both concentration and time dependent (Savage B., Bottini E. & Ruggeri Z M., “Interaction of integrin alpha IIb beta with multiple fibrinogen domains during platelet adhesion”, J. Biol. Chem. 270: 28812–7 (1995)). Isolated constituent chains of fibrinogen (Aα1, Aα2 and Bβ) released upon activation of the fibrinogen by thrombin were observed to stimulate fibroblast proliferation by 23–31% above controls whereas isolated γ chain had no effect (Gray. A. J., Bishop, J. E., Reeves, J. T. and Laurent, G. J.; “Aα and Bβ Chains of fibrinogen stimulate proliferation of human fibroblasts”, J. Cell Sci., 104: 409–413, (1993)). Human polymorphonuclear leukocytes (PMN) were shown to bind to fibrin(ogen) coated surfaces via a type 3 (CD11b/CD18) complement receptor homologous to the GPIIb/IIIa receptor through a decamer of the γ chain carboxy terminus (LGGAKQAGDV). Vasoactive peptides corresponding to residues 43–47 of the Bβ chain and 220–230 of the Aα chain were identified (Gray A. J., Bishop, J. E., Reeves, J. T. and Laurent, G. J.; “Aα and Bβ Chains of fibrinogen stimulate proliferation of human fibroblasts”, J. Cell Sci., 104: 409–413, (1993)).
The biological activities of only few other fibrinogen breakdown products have been investigated, whose cellular activity seems to be widely variable (Saldeen T: Vasoactive peptides derived from degradation of Fibrinogen and fibrin. Proc. NY Acad Sci USA, 408: 424–431 (1983)).
Fibrinogen itself when bound to sepharose beads, did not significantly affect cell proliferation, but elicited haplotactic/attachment reactions from human (HF) or mouse (MF) fibroblasts, endothelial cells (EC) and smooth muscle (SMC) cells. Thrombin treatment of fibrinogen-sepharose beads (SB-fib), which would not affect the carboxy termini of the molecule, did not alter cellular responses, though plasmin, which clips off gamma carboxy termini and digests D-domain sequences, nearly totally abrogated the cell-attractant properties of SB-fib (Gorodetsky R., Vexler A., An J., Mou X, Marx G. (1998) J. Lab. Clin. Med. 131: 269–280).
Specific epitopes on fibrinogen have been hypothesized to express cell binding (haptotactic) properties. However, the amino acid sequence(s) of such putative haptotactic epitopes have not yet been specified. The identification of such epitopes would have a number of applications, enabling more specific intervention in the wound healing process and in the development of novel therapeutic compositions or devices. Furthermore, novel diagnostic tests for testing cellular haptotactic responses could potentially be developed. Thus, the identification of these specific epitopes or peptides exhibiting cellular activity would have great utility.
There is thus a recognized need for, and it would be highly advantageous to have, a peptide or peptides with specifically determined cellular effects, such as cell proliferative or chemotactic or haptotactic properties, which do not require the presence of the entirety of the fibrin molecule to exert cellular effects.